Cytokine receptor modulators, method of identifying same, and method of modulating cytokine receptors activity with same

ABSTRACT

The present invention relates to a method for identifying a non-competitive peptide, which inhibits the activity of a cytokine receptor. This method includes the steps of selecting a candidate peptide containing from about 7 to about 20 amino acids derived from a flexible region of a cytokine receptor, and determining the ability of the peptide to inhibit or promote the oligomerization and/or activation of the receptor by measuring an activity of the receptor in the absence or the presence of the candidate peptide, wherein the non-competitive peptide is selected when the activity of the receptor is measurably lower in the presence of the peptide as compared to in the absence of the peptide so identified. This invention also provides agonists of cytokine receptor activity. Pharmaceutical compositions that comprise the identified peptides are disclosed. Also disclosed are methods for treating patients with a disease or condition associated with abnormal cytokine receptor mediated function or activity such as inflammatory, autoimmune and vascular diseases.

This application claims the benefit of U.S. Provisional Application No.60/420,679, filed Oct. 24, 2002, and U.S. Provisional Application No.60/423,530, filed Nov. 5, 2002. The entire text of the above provisionalapplications are specifically incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to cytokine receptor agonists andantagonists, to a method of identifying same, and to a method ofmodulating cytokine receptors activity with same. More specifically, thepresent invention is concerned with extracellular, non-competitivecytokine receptor modulators, a method of identifying same, theiridentification and their uses. More particularly, the present inventionis concerned with extracellular, non-competitive cytokine receptorantagonists, a method of identifying same, their identification andtheir therapeutic uses.

BACKGROUND OF THE INVENTION

Cytokines are generic terms for designating biologically activehormone-like proteins (interleukins, interferons, tumor necrosis factor,growth factors) that mediate their effects through a superfamily ofreceptors. Cytokines and their receptors constitute a powerful controlnetwork by which cells signal and coordinate cell proliferation anddifferentiation, cell death and survival. Cytokines are low molecularweight peptides having very potent biological activity. Their mechanismof action is generally autocrine and paracrine and act by ultimatelyregulating gene expression.

Cytokines and their receptors are thus implicated in major diseases.They regulate hematopoiesis, immunity and development of the nervoussystem. Most of all, they contribute to the development of afflictionssuch as cancer, inflammatory and autoimmune reactions, asthma, allergy,thrombosis, vascular diseases and septic shock by influencing aberrantor overexpressing genes leading to diseases. Cytokines and growthfactors mediate tightly regulated biological effects in order to ensureproper control and functioning of the immune system. Therefore,cytokines are also involved in pathological conditions such asinflammation (e.g. rhumatoid arthritis) and tissue degeneration.Diseases which may develop or progress as a result of defects incytokine or growth factor mediated cell signaling have a high prevalencein the population and are associated with significant morbidity and/ormortality. For these reasons cytokine receptors are importanttherapeutic target.

The treatments available for these pathologies are currently limited.They often result in high toxicity and secondary effects. The demand inthe medical world for safer and more targeted therapies is thereforeconsiderable.

The current approaches in the field of cytokines antagonists include thedevelopment of soluble receptors, monoclonal antibodies directed againstcytokines, mimetics of cytokines, antisense techniques and kinasesinhibitors. Few of these strategies have been successful in drugdevelopment, however. Nevertheless, certain antibodies targeting theligand and the receptor, natural soluble receptor inhibitors (eg.IL1ra), and decoy soluble receptors have displayed interesting results.For instance, Trastuzumab (Herceptin, Roche) a monoclonal antibody whichbinds the HER-2/neu protein tyrosine kinase, and ZD1839 (Iressa,Astra-Zeneca), a small molecule which binds the EGF receptor are eitherin clinical trials or available for the treatment of certain diseases.

Non competitive antagonists of cytokines have also been described. Ininternational application no. WO 93/14781 published in 1993, Foxdescribes the use of non-competitive peptides targeting intracellulardomains of EGF. Intracellular domains are difficult to reach by peptidesbecause of the barrier that the cell membrane constitutes.

Antagonists of the prior art are thus either competitive (e.g. solublereceptors, antibodies, cytokine mimetics), not very selective (e.g.tyrosine kinase inhibitors), costly to produce or difficult to apply invivo (e.g. antisense). Because the ligand exceeds by far theconcentration of the receptor, the concentration of competitiveantagonists needed to inhibit the receptor is often substantial.

There is therefore a need for non-competitive, selective, extracellularand simple to identify, select and produce antagonists of cytokines.

The present invention seeks to meet these needs and other needs.

The present description refers to a number of documents, the content ofwhich is herein incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

The present invention thus concerns non-competitive and selectiveextracellular cytokine receptor modulators, and methods of selecting andof using same.

The peptides, derivatives and peptidomimetics thereof of the presentinvention are derived from selected cytokine receptor flexible regions.Cytokine receptor agonists or antagonists of the present inventionpossess a unique mechanism and site of action for inhibiting cytokinereceptors activity. They are peptides strategically positioned on atleast one of an extracellular flexible region including juxtamembranousregions, flexible regions between domains of the cytokine receptor, andoligomerization site, that are important for the appropriateconformation of the receptor which enables signaling. In one embodimentthe flexible region is required for proper oligomerization to occur. Insuch an embodiment, appropriate conformation of the receptor is neededto allow adequate positioning of the protein chains to enableoligomerization of the receptor and its resulting activation.

Cytokine receptors subfragments or peptides of this invention maypromote or stabilize a particular conformation of the cytokine receptorwhich results in inhibition or activation of the receptor activity. Inparticular, the antagonists of this invention do not necessarilyinterfere directly with the oligomerization site. They may, for example,exert their antagonistic activity by directly or indirectly preventingthe oligomerization of the complementary protein chains (of homodimersas well as heterodimers receptors) of the extracellular domain of thecytokine receptor. This process effectively prevents activation of theintracellular receptor domains responsible for cytokine enzymaticfunction. Subsequent cell transduction events leading to overexpressionof the ligand and/or cell bound receptors responsible in part fordisease expression are thereby prevented.

In the alternative, one can use cytokine receptors subfragment peptidesor derivatives to promote or stabilize the active cytokine receptorstructure capable signal transduction. Such peptides are consideredagonists of the present invention. Cytokine receptor modulators of thepresent invention possess a number of advantages over the prior art.

Because they have extracellular targets, unlike certain known drugcandidates which target intracellular regions of the cytokine receptors,the antagonists of the present invention do not necessitate a priorpermeabilization or other disruption of cell membranes to gain access tothe target in order to produce a pharmacological response.

Because they are non competitive, a smaller amount of the antagonists ofthe present invention is necessary to inhibit the receptor that theytarget, as compared to competitive inhibitors.

As peptides, the antagonists of the present invention are advantageouslysimple to synthesize.

In order to provide a clear and consistent understanding of terms usedin the present description, a number of definitions are provided hereinbelow.

In view of the importance of the function of cytokine receptors innumerous pathway and conditions in animals, the present invention hasbroad impact on the screening, identification, validation and treatmentof conditions or diseases associated with abnormal functioning of thesecytokine receptors.

Unless defined otherwise, the scientific and technological terms andnomenclature used herein have the same meaning as commonly understood bya person of ordinary skill to which this invention pertains. Generally,the procedures for cell cultures, infection, molecular biology methodsand the like are common methods used in the art. Such standardtechniques can be found in reference manuals such as for exampleSambrook et al. (1989, Molecular Cloning—A Laboratory Manual, ColdSpring Harbor Laboratories) and Ausubel et al. (1994, Current Protocolsin Molecular Biology, Wiley, New York).

Cytokine Receptors

The term “cytokine” refers herein to any cytokine including growthfactor. Similarly, the term “cytokine receptors” refers herein to anycytokine receptor including growth factor receptors. The cytokinereceptors comprise a number of families including 1-tyrosine kinasesreceptors, such as vascular endothelial growth factor receptors (VEGFR),PDGFR, IGF-1R, FGFR, EGFR; 2-type I receptors, such as Interleukins-2,3, 4, 5, 7, 9 and 15; 3-type II receptors, such as interleukins 10,IFNαR, IFNβR, IFNR; 4-TGFβ; 5-chemokines; and 6-NGF/TNF;6-interleukins-1 types I and II. The present invention encompassespeptidic agonists or antagonists directed at any cytokine.

The method of identifying cytokines antagonists of the present inventionis based on the localization of flexible extracellular regions,including regions between domains, long loops between two f chains, aswell as juxtamembranous regions of the receptor, which are important forthe appropriate conformation and/or oligomerization of the subunits ofthe receptor and/or its resulting activation. These regions can bedetermined using for example crystallography data, model structures,data bases, sequence alignments and the like. For example, the targetedregions were established herein based on crystal structure data providedby crystallography for IL-1R and IGF-1R and on published model structurefor IL-4R. Databases such as Swiss-Prot and NCBI as well as sequencesalignments with CLUSTALW and MOTIFSCAN enabled a comparison between manyregions constituting the receptors domains and their structuralsimilarities with flexible regions of the vascular endothelial growthfactor receptor (VEGFR). It should be noted that the flexible regions ofthe present invention need not be directly involved in oligomerization.Indeed, regions which facilitate oligomerization or regions that areimplicated in conformational changes needed for receptor signaling arealso within the scope of the present invention. The same principle applyto the identification of cytokine agonists.

The terminology “juxtamembranous region of a receptor” refers herein toan extracellular region of the receptor located in the vicinity of thecellular membrane. More particularly in a region which spans a length ofup to about 20 amino acids.

The terminology “flexible region of a receptor” refers herein to anyregion of the receptor that possesses sufficient flexibility to enablethis region to bend, extend, twist or otherwise change its conformationand by which conformational change alone or in combination with otherconformational changes of other flexible regions, receptor's activity isinduced or facilitated. It includes juxtamembranous regions,oligomerization regions including those having secondary structures suchas α helix, β sheet, loops, β turns, and flexible regions betweendomains of the receptor or in long loops between two β chains.

Peptides Preparation

The peptides of this invention, including the analogs and other modifiedvariants, may generally be synthesized according to the FMOC protocol inan organic phase with protective groups. They can be purified with ayield of 70% with HPLC on a C18 column and eluted with an acetonitrilegradient of 10-60%. Their molecular weight can then be verified by massspectrometry.

The peptides of the invention may also be prepared according to thesolid phase synthetic method. For example, the solid phase synthesis iswell known and is a common method for preparation of peptides, as are avariety of modifications of that technique [Merrifield (1964), J. Am.Chem. Soc., 85: 2149; Stewart and Young (1984), Solid Phase PeptideSynthesis, Pierce Chemical Company, Rockford, Ill.; Bodansky andBodanszky (1984), The Practice of Peptide Synthesis, Springer-Verlag,New York; Atherton and Sheppard (1989), Solid Phase Peptide Synthesis: APractical Approach, IRL Press, New York].

Alternatively, peptides of this invention may be prepared in recombinantsystems using polynucleotide sequences encoding the peptides. It isunderstood that a peptide of this invention may contain more than one ofthe above described modifications within the same peptide. Also includedin this invention are pharmaceutically acceptable salt complexes of thepeptides of this invention or their derivatives.

Peptides

Peptides of the present invention may therefore be constituted solely ofL-amino acid sequences identical to amino acid sequences of flexibleregions of an animal cytokine receptor and preferably of the humancytokines receptor that they target (subfragment peptides) and anymutated peptide that can be generated.

While subfragment peptides are effective in inhibiting wild-typecytokines in vitro, their effectiveness in vivo might be compromised bythe presence of proteases. Serum proteases have specific substraterequirements. The substrate must have both L-amino acids and peptidebonds for cleavage. Furthermore, exopeptidases, which represent the mostprominent component of the protease activity in serum, usually act onthe first peptide bond of the peptide and require a free N-terminus(Power, et al. (1993), Pharmaceutical Res., 10:1268-1273). In light ofthis, it is often advantageous to utilize modified versions ofsubfragment peptides. The modified peptides retain the structuralcharacteristics of the original L-amino acid peptides that conferbiological activity with regard to cytokines, but are advantageously notreadily susceptible to cleavage by proteases and/or exopeptidases.

Therefore, in specific modes, the peptides of this invention are notsubfragment peptides, although their amino acid sequence is derived fromthe linear sequence of the human cytokine receptors or the correspondingsequences of non-human cytokine receptors and able to inhibit a cytokinereceptor's activation (e.g. oligomerization) and more particularly theactivation of a human cytokine receptor. Particularly, suitablenon-human cytokine receptors sources include mouse, rat, quail andhorse. It is thus apparent that multiple systems can provide suitablepeptides and derivatives from which the cytokine receptor antagonists ofthe present invention can be derived.

The term “peptides” as referred to herein therefore includes cytokinereceptor subfragment peptides, D-peptides and other modified forms ofthe peptides, so long as the modification does not alter ability tomodulate cytokine receptor activity. All agonists and antagonistspeptides of this invention share the ability to modulate the activity ofspecific cytokines receptors. Non-limiting examples of modificationsinclude N-terminal acetylation, glycosylation, and biotinylation.Particular modified versions of the subfragment peptides according tothe present invention are further described below. Although the peptidesof the present invention encompass any peptide derived from the flexibleregions of cytokines receptors, preferred peptides of the presentinvention are chosen so as to be specific to a particular receptorisoform (e.g. VEGFR-2) to ensure that their spatial conformation iscomplementary to the flexible region that they target. This lattercharacteristic is obtained by choosing where the peptide will be cutaccording to the properties afforded by each amino-acid in the remainingsequence (e.g. if the peptide has to follow the specific curve of thedomain targeted).

The term “peptides derived from a flexible region” refers herein topeptides of 5 to about 20 amino acids that have been generated tocorrespond to segments of 5 to 20 contiguous amino acids locatedanywhere in the flexible regions and that may have been subjected tofurther modification or functional derivation as described herein.Preferably, the peptides derived from a flexible region is a peptide ofat least 7 amino acids.

D-amino acid peptides can have modifications at the N-terminalamino-acid and at the C-terminal amino-acid. The presence of anN-terminal or C-terminal D-amino acid increases the serum stability of apeptide which otherwise contains L-amino acids, because exopeptidasesacting on these residues cannot utilize a D-amino acid as a substrate(Powell, et al. (1993)). Cyclic peptides have no free N- or C-termini.Thus, they are not susceptible to proteolysis by exopeptidases, althoughthey are of course susceptible to endopeptidases, which do not cleave atpeptide termini. Thus, the amino acid sequences of the peptides withN-terminal or C-terminal D-amino acids and of the cyclic peptides areusually identical to the sequences of the subfragment peptides to whichthey correspond, except for the presence of an N-terminal or C-terminalD-amino acid residue, or their circular structure, respectively.

Substitution of unnatural amino acids for natural amino acids in asubsequence of the subfragment of cytokine receptor peptide can alsoconfer resistance to proteolysis. Such a substitution can, for instance,confer resistance to proteolysis by exopeptidases acting on theN-terminus. Such substitutions have been described (Coller, et al.(1993), J. Biol. Chem., 268:20741-20743, incorporated herein byreference) and these substitutions do not affect biological activity.Furthermore, the synthesis of peptides with unnatural amino acids isroutine and known in the art (see, for example, Coller, et al. (1993),supra).

An other effective approach to confer resistance to peptidases acting onthe N-terminal or C-terminal residues of a peptide is to add chemicalgroups at the peptide termini, such that the modified peptide is nolonger a substrate for the peptidase. One such chemical modification isglycosylation of the peptides at either or both termini. Certainchemical modifications, in particular N-terminal glycosylation, havebeen shown to increase the stability of peptides in human serum [Powellet al. (1993), supra]. Other chemical modifications which enhance serumstability include, but are not limited to, the addition of an N-terminalalkyl group, consisting of a lower alkyl of from 1 to 20 carbons, suchas an acetyl group, and/or the addition of a C-terminal amide orsubstituted amide group. In particular the present invention includesmodified peptides consisting of subfragment peptides bearing anN-terminal acetyl group and a C-terminal amide group.

Longer peptide sequences which result from the addition of extra aminoacid residues to the peptides of the invention are encompassed in thepresent invention since they should have the same biological activity(inhibit oligomerization of cytokines) as the peptides described above.While peptides having a substantial number of additional amino acids arenot excluded, it will be recognized that some large polypeptides mayassume a configuration that masks the effective sequence, therebypreventing binding to cytokines. These derivatives will act ascompetitive antagonists and are thereby excluded from the invention.Thus, while the present invention encompasses peptides or derivativeshaving an extension, such longer peptides should be selected as notdestroying the modulating activity of the peptide or derivative.

The present invention also encompasses peptides constituted of thesequences of two peptides having separately the property of inhibitingthe activation (e.g. oligomerization) of a particular cytokine receptor,but not being contiguous within the flexibility regions. These peptidescan also be described as having a sequence corresponding to theparticular cytokine receptor with an internal deletion.

In another embodiment of this invention the peptides are reverse-Dpeptides corresponding to the amino acid sequence of the cytokine. Theterm “reverse-D peptide” refers herein to peptides containing D-aminoacids, arranged in a reverse sequence relative to a peptide containingL-amino acids. Thus, the C-terminal residue of an L-amino acid peptidebecomes N-terminal for the D-amino acid peptide, and so forth. Forexample, the sequence of the reverse-D peptide corresponding tosubfragment peptide SEQ ID NO: 1 is: GVLIIIELNTKEQA. Reverse-D peptidesretain the same tertiary conformation, and therefore the same activity,as the L-amino acid peptides, but are more stable to enzymaticdegradation in vitro and in vivo, and thus have greater therapeuticefficacy than the original peptide (Brady and Dodson (1994), Nature,368: 692-693; Jameson et al. (1994), Nature, 368: 744-746).

As used herein, the designation “functional derivative” denotes, in thecontext of a functional derivative of an amino acid sequence, a moleculethat retains a biological activity (either function or structural) thatis substantially similar to that of the original sequence. Thisfunctional derivative or equivalent may be a natural derivative or maybe prepared synthetically. Such derivatives include amino acid sequenceshaving substitutions, deletions, or additions of one or more aminoacids, provided that the biological activity of the protein is conserved(e.g. it acts as a non-competitive inhibitor or agonist of a cytokinereceptor). The substituting amino acid generally has chemico-physicalproperties which are similar to that of the substituted amino acid. Thesimilar chemico-physical properties include, similarities in charge,bulkiness, hydrophobicity, hydrophylicity and the like. The term“functional derivatives” is intended to include “segments”, “variants”,“analogs” or “chemical derivatives” of the subject matter of the presentinvention.

Thus, the term “variant” refers herein to a protein which issubstantially similar in structure and biological activity to theprotein or nucleic acid of the present invention.

The functional derivatives of the present invention can be synthesizedchemically or produced through recombinant DNA technology. All thesemethods are well known in the art.

While peptides of specific embodiments of the present invention arepreferably derived from human cytokines receptors, the invention shouldnot be so limited. Indeed, in view of the significant conservation offlexible regions of these genes throughout evolution, sequences fromdifferent species, as discussed above and preferably mammalian species,could be used in the assays of the present invention. For instance,non-limiting examples for the VEGFR protein are the quail, mouse, ratand horse VEGFR protein sequences which show 70%, 82% and 82%similarity, respectively with the human VEGFR protein sequence.Similarly, the IL-1R mouse, rat and horse protein sequences show a 68%,67% and 77% sequence similarity, respectively. Also, the IL-4R mouse andhorse protein sequences show a 48% and 59% sequence similarity,respectively (as calculated by Blast™).

For administration to humans, the prescribing medical professional willultimately determine the appropriate form and dosage for a givenpatient, and this can be expected to vary according to the chosentherapeutic regimen (e.g. peptides, variants, mimetics), the responseand condition of the patient as well as the severity of the disease.

Composition within the scope of the present invention should contain theactive agent (e.g. peptide) in an amount effective to achieve thedesired therapeutic effect while avoiding adverse side effects.Typically, the nucleic acids in accordance with the present inventioncan be administered to mammals (e.g. humans) in doses ranging from 0.005to 1 mg per kg of body weight per day of the mammal which is treated.Pharmaceutically acceptable preparations and salts of the active agentare within the scope of the present invention and are well known in theart (Remington's Pharmaceutical Science, 16th Ed., Mack Ed.). For theadministration of polypeptides, antagonists, agonists and the like, theamount administered should be chosen so as to avoid adverse sideeffects. The dosage will be adapted by the clinician in accordance withconventional factors such as the extent of the disease and differentparameters from the patient. Typically, 0.001 to 50 mg/kg/day will beadministered to the mammal.

Assays to Identify Peptides of the Present Invention

Preferred methods for testing the ability of candidate compounds toinhibit the various cytokine receptors activity are presented herein. Itwill be understood that the invention is not so limited. Indeed, oftenassays well known in the art can be used in order to identifynon-competitive, extracellular agonists or antagonists of the presentinvention.

As used herein, “cytokine receptor activity or activation” refers to anydetectable biological activity of these proteins. This includes anyphysiological function attributable to a cytokine receptor such as anystandard biochemical measurement of these receptors, conformationalchanges, phosphorylation status, any downstream effect of the receptor'ssignaling such as protein phosphorylation, kinase effect or any otherfeature of the protein that can be measured with techniques known in theart. Measuring the effect of a candidate peptide on its ability tomodulate the oligomerization of the receptor is measuring a cytokinereceptor's activity according to this invention. Broadly intra- orinter-molecular binding of the receptor in the absence vs the presenceof the peptide of the invention is yet another example of a biologicalactivity according to the invention.

The assays of this invention employ either a natural or recombinantcytokine receptor. A cell fraction or cell free screening assays forinhibitors of cytokine receptor activity can use in situ purified, orpurified recombinant cytokine receptor. Cell-based assays can employcells which express cytokine receptor naturally, or which containrecombinant cytokine receptor. In all cases, the biological activity ofcytokine receptor can be directly or indirectly measured; thusinhibitors or activators of cytokine receptor activity can beidentified. The inhibitors or activators themselves may be furthermodified by standard combinatorial chemistry techniques to provideimproved analogs of the originally identified compounds.

It shall be understood that the “in vivo” experimental model can also beused to carry out an “in vitro” assay.

In Vitro Assays

In one embodiment, candidate peptides are tested for their ability toactivate or inhibit cytokine receptor's ability to modulate cellularproliferation with the incorporated triated thymidine method. In yetother preferred embodiments, candidate peptides are tested for theirability to inhibit a particular cytokine receptors ability to modulatecellular proliferation, using for example, the assays described in BakerF. L. et al. (1995) Cell Prolif. 28(1):1-15; Cheviron N. et al. (1996)Cell Prolif. 29(8):43746; Hu Z. W. et al. (1999) J: Pharmacol. Exp.Ther. 290(1):28-37; and Elliott K. et al. (1999) Oncogene18(24):3564-73.

In another preferred embodiment, candidate peptides are tested for theirability to modulate the phosphorylation state of cytokine protein orportion thereof, or an upstream or downstream target protein, using forexample an in vitro kinase assay. Briefly, a cytokine receptor targetmolecule (e.g. an immunoprecipitated receptor from a cell lineexpressing such a molecule), can be incubated with radioactive ATP,e.g., [gamma-³²P]-ATP, in a buffer containing MgCl² and MnCl², e.g., 10mM MgCl² and 5 mM MnCl². Following the incubation, theimmunoprecipitated receptor target molecule, can be separated bySDS-polyacrylamide gel electrophoresis under reducing conditions,transferred to a membrane, e.g., a PVDF membrane, and autoradiographed.The appearance of detectable bands on the autoradiograph indicates thatthe receptor substrate has been phosphorylated. Phosphoaminoacidanalysis of the phosphorylated substrate can also be performed in orderto determine which residues on the receptor substrate arephosphorylated. Briefly, the radiophosphorylated protein band can beexcised from the SDS gel and subjected to partial acid hydrolysis. Theproducts can then be separated by one-dimensional electrophoresis andanalyzed on, for example, a phosphoimager and compared toninhydrin-stained phosphoaminoacid standards. Assays such as thosedescribed in, for example, Tamaskovic R. et al. (1999) Biol. Chem.380(5):569-78.

In other embodiments, candidate peptides targeting IL-1R are tested withPGE₂ levels, IL-6, collagenase expression in chondrocytes and RPE;candidate peptides targeting IGF-1R are tested with Akt in Du145 andPC12; candidate peptides targeting IL-4R are tested with Akt in Thelperand PAEC and with VCAM-1 expression in PAEC.

In Vivo Assays

The assays described above may be used as initial or primary screens todetect promising lead compounds for further development. Lead peptideswill be further assessed in additional, different screens. Therefore,this invention also includes secondary cytokine receptors screens whichmay involve various assays utilizing mammalian cell lines expressingthese receptors or other assays.

Tertiary screens may involve the study of the identified inhibitors inanimal models for clinical symptoms. Accordingly, it is within the scopeof this invention to further use an agent (peptide or peptidomimetic)identified as described herein in an appropriate animal model such as arat or a mouse. For example, a peptide can be used in an animal model todetermine the efficacy, toxicity, or side effects of treatment with suchan agent. Alternatively, an agent identified as described herein can beused in an animal model to determine the mechanism of action of such anagent. Furthermore, this invention pertains to uses of novel agentsidentified by the above-described screening assays for treatment (e.g.treatments of different types of disorders associated with aderegulation or malfunction of a cytokine receptor), as describedherein. Preferred such experiments include collagen-induced arthritis inrat, acute septic shock in rat, tumor growth in immunosuppressed mouse,sensitization of the airways in newborn mice and any other known animalmodel including transgenics.

Assays to Identify Peptidomimetics

Non-peptidyl compounds generated to replicate the backbone geometry andpharmacophore display (peptidomimetics) of the peptides identified bythe methods of the present invention often possess attributes of greatermetabolic stability, higher potency, longer duration of action andbetter bioavailability.

The peptidomimetics compounds of the present invention can be obtainedusing any of the numerous approaches in combinatorial library methodsknown in the art, including: biological libraries; spatially addressableparallel solid phase or solution phase libraries; synthetic librarymethods requiring deconvolution; the ‘one-bead one-compound’ librarymethod; and synthetic library methods using affinity chromatographyselection. The biological library approach is limited to peptidelibraries, while the other four approaches are applicable to peptide,non-peptide oligomer or small molecule libraries of compounds (Lam,Anticancer Drug Des. 12: 145, 1997). Examples of methods for thesynthesis of molecular libraries can be found in the art, for examplein: DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA. 90:6909; Erb et al.(1994) Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann et al. (1994), J.Med. Chem. 37:2678; Cho et al. (1993) Science 261:1303; Carrell et al.(1994) Angew. Chem, Int. Ed Engl. 33:2059; and ibid 2061; and in Gallopet al. (1994). Med. Chem. 37:1233. Libraries of compounds may bepresented in solution (e.g. Houghten (1992) Biotechniques 13:412-421) oron beads (Lam (1991) Nature 354:82-84), chips (Fodor (1993) Nature364:555-556), bacteria or spores (Ladner U.S. Pat. No. 5,223,409),plasmids (Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or onphage (Scott and Smith (1990); Science 249:386-390). Examples of methodsfor the synthesis of molecular libraries can be found in the art, forexample in: DeWitt et al. (1993) supra; Erb et al. (1994) supra;Zuckermann et al. (1994) supra; Cho et al. (1993) supra; Carrell et al.(1994) supra, or luciferase, and the enzymatic label detected bydetermination of conversion of an appropriate substrate to product.

In one embodiment, the peptidomimetics compounds of the presentinvention are preferably obtained with the following three phaseprocess. 1) Scanning the peptides of the present invention to identifyregions of secondary structure necessary for recognition and activitytoward the cytokine receptor; 2) use conformationally constraineddipeptide surrogates to refine the backbone geometry and provide organicplatforms corresponding to these surrogates; 3) Use the best organicplatforms to display organic pharmocophores in libraries of candidatesdesigned to mimic the desired activity of the native peptide. In moredetails the three phases are as follows.

In phase 1, the peptide leads are scanned and their structure abridgedto identify the requirements for their activity. A series of peptideanalogs of the original are synthesized. In phase 2, the best peptideanalogs are investigated using the conformationally constraineddipeptide surrogates. Indolizidin-2-one, indolizidin-9-one andquinolizidinone amino acids (I²aa, I⁹aa and Qaa respectively)) are usedas platforms for studying backbone geometry of the best peptidecandidates. These platforms are introduced at specific regions of thepeptide in order to orient the pharmacophores in different directions.Biological evaluation of these analogs identifies improved leads thatmimic the geometric requirements for activity. In phase 3, the platformsfrom the most active leads are used to display organic surrogates of thepharmacophores responsible for activity of the native peptide. Thepharmacophores and scaffolds are combined in a parallel synthesisformat.

In summary, based on the disclosure herein, those skilled in the art candevelop peptides and peptidomimetics screening assays which are usefulfor identifying compounds which are useful for inhibiting cytokinereceptors. Compounds so identified might also be shown to activate thesereceptors. The assays of this invention may be developed forlow-throughput, high-throughput, or ultra-high throughput screeningformats. Of course, assays of the present invention include assays whichare amenable to automation.

More specifically, in accordance with one embodiment, the presentinvention, there is provided a method for identifying a non-competitivepeptide which inhibits the oligomerization of a cytokine receptor, themethod comprising the steps of selecting a candidate peptide containingfrom about 7 to about 20 amino acids derived from a flexible region ofthe receptor, and determining the ability of the peptide to inhibit theoligomerization of the receptor by measuring an activity of the receptorin the presence of a compound known to activate the receptor and in theabsence or the presence of the candidate peptide, wherein thenon-competitive peptide is selected when the activity of the receptor ismeasurably lower in the presence of the peptide as compared to in theabsence thereof.

There is also provided a non-competitive extracellular cytokine receptorantagonist wherein the antagonist is a peptide containing from about 7to about 20 amino-acids derived from a flexible region of the cytokinereceptor.

The present invention also provides methods of treating diseases orconditions associated with a abnormal activity of a cytokine receptorcomprising administration of a suitable amount of peptide or derivativeof the invention.

The present invention also relates to pharmaceutical compositionscomprising a modulating amount a or cytokine receptor subfragmentpeptide or derivative of the present invention, together with a suitablepharmacological carrier.

The terms “inhibiting,” “reducing” or “prevention,” or any variation ofthese terms, when used in the claims and/or the specification includesany measurable decrease or complete inhibition to achieve a desiredresult.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

It is contemplated that any embodiment discussed in this specificationcan be implemented with respect to any method or composition of theinvention, and vice versa. Furthermore, compositions and kits of theinvention can be used to achieve methods of the invention.

Throughout this application, the term “about” is used to indicate that avalue includes the standard deviation of error for the device or methodbeing employed to determine the value.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.”

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

In the appended drawings:

FIG. 1 schematically illustrates the position of VEGFR antagonists onthe receptor according to specific embodiments of the present invention;

FIG. 2 graphically illustrates in panel A results of proliferation assayin porcine microvascular endothelial cells in presence of VEGF (2 ng/ml)and peptides 2.1, 2.2, 2.3 (10 μM). In panel B are graphicallyillustrated dose-response of peptides in pulmonary arterial endothelialcells (PAEC) in presence of VEGF (2 ng/ml) and increasing doses ofpeptides. In panel C is graphically illustrated the effect ofintravitreally injected peptides (10 μM [estimated final intraocularconcentration]) of the present invention on neovascularization in ratretinas exposed to hyperoxic conditions;

FIG. 3 shows the sequence of the human VEGFR-2 (Flk-1). Boxed orunderlined sequences represent the identified flexible region of VEGFR;

FIG. 4 shows the sequence of human Interleukin-1 receptor (IL-1R-alpha).Boxed or underlined sequences represent the identified flexible regionof IL-1R-alpha;

FIG. 5 shows the sequence of human Interleukin-1 receptor accessoryprotein (IL-1 RacP). Boxed or underlined sequences represent theidentified flexible region of IL-1 RacP;

FIG. 6 shows the sequence of human Insulin-like growth factor I receptor(IGF-1R). Boxed or underlined sequences represent the identifiedflexible region of; IGF-1R;

FIG. 7 shows the sequence of the human alpha chain of the Interleukin 4receptor (IL-4R). Boxed or underlined sequences represent the identifiedflexible region of; (IL-4R).

FIG. 8 graphically illustrates results of proliferation assays incarcinome A549 cells in presence of IGF-1 (10 ng/ml-Panel A) (1ng/ml-Panel B) and various concentrations of the peptides APG-201,APG-202 and APG-204;

FIG. 9 graphically illustrates results of proliferation assays incarcinome A549 cells in presence of IL-1 (10 ng/ml-Panel A) (1ng/ml-Panel B) and various concentrations of the peptides API-101,API-103 and API-106;

FIG. 10 graphically illustrates results of proliferation assays incarcinome A549 cells in presence of IL-4 (1 ng/ml) and variousconcentrations of the peptides API-401, API-402, API-403, API-404 andAPI-405;

FIG. 11 shows an alignment of the human IL-1R sequence withcorresponding mouse, rat and horse sequences;

FIG. 12 shows an alignment of the human IL-4R sequence withcorresponding mouse and horse sequences; and

FIG. 13 shows an alignment of the human VEGFR2 sequence withcorresponding mouse, rat and quail sequences.

DESCRIPTION OF SPECIFIC EMBODIMENTS

Table 1 presents the localization of flexible regions of variousrepresentative members of the cytokine receptors families along withexemplary peptide sequences derived from these regions and chosen fortheir specificity to the particular member they target. As explainedabove, many peptides can be derived from the targeted regions of thepresent invention and the peptides described hereinbelow are onlyexemplary.

TABLE 1 Amino acids involved in the oligomerization and stability ofreceptors of representative members of various cytokine receptorsLOCALISATION OF THE SEQUENCE CYTOKINES FROM THE RECEPTOR SPECIFICREGIONS STARTING TYPES RECEPTORS TARGETED METHIONINE PEPTIDE SEQUENCESTyrosine VEGFR2 Juxtamembranous Aa 745-770 AQEKTNLEIIILVG (2.1) Kinase(Flk-1) SEQ ID NO. 1 receptor Ig3-Ig4 Aa 320-350 EATVGERVRL (2.2) SEQ IDNO. 2 Ig-4 dimerization Aa 350-400 LPLESNHTLK (2.3) domain SEQ ID NO. 3Ig-4-Ig-5 Aa 400-440 SPVDSYQYGTT; SEQ ID NO. 4 VILTNPISKE; SEQ ID NO. 5Ig-5-Ig 6 Aa 481-565 NKVGRGERVI; SEQ ID NO. 6 MPPTEQESV SEQ ID NO. 7Ig-6-Ig-7 Aa 640-685 RKTKKRHCV; SEQ ID NO. 8 TVLERVAPT; SEQ ID NO. 9TSIGESIEV SEQ ID NO. 10 IGF-1R On chain α: Juxtamembranous Aa 725-740SIFVPRPERK; SEQ ID NO. 11 NFLHNSIFV; SEQ ID NO. 12 Cyst rich domain-L2Aa 320-335 EGPCPKVCE; SEQ ID NO. 13 L2-FbnIII-1 Aa 487-527 ESDVLHFTST;SEQ ID NO. 14 FbnIII-1-FbnIII2a Aa 595-620 RTNASVPSI; SEQ ID NO. 15FbnIII-2a-Insert Aa 660-690 IRKYADGTI; domain SEQ ID NO. 16 On chain β:Insert domain- Aa 780-799 ENFIHLIIA; FbnIII2b SEQ ID NO. 17 AKTGYENFIH;SEQ ID NO. 18 FbnIII2b-FbnIII3 Aa 820-840 KERTVISNLR; SEQ ID NO. 19Juxtamembranous Aa 917-947 FVFARTMPA; SEQ ID NO. 30 EGFR JuxtamembranousAa 640-650 NGPKIPSIAT; SEQ ID NO. 31 Loop L2-S2 Aa 495-515 ATGQVCHAL;(flexible) SEQ ID NO. 32 Loop S1-L2 (Hinge) Aa 335-345 RKVCNGIGIGE; SEQID NO. 33 Type I: IL-4R Juxtamembranous Aa 210-240 WHNSYREPF; ChainγcSEQ ID NO. 34 YREPFEQHLL SEQ ID NO. 35 Hinge zone D2 Aa 125-216SDTLLLTWS; SEQ ID NO. 36 IYNVTYLE; SEQ ID NO. 37 IAASTLKSGIS; SEQ ID NO.38 Loop D1-D2 Aa 112-125 KPSEHVKPR; SEQ ID NO. 39 Single GHRJuxtamembranous Aa 250-270 FTCEEDFYFPW; chain flexible region Aa 160-240SEQ ID NO. 40 (D1-D2) SVDEIVQPD; SEQ ID NO. 41 MDPIDTTSVPVY; SEQ ID NO.42 IL-1R IL-1R Juxtamembranous Aa 320-341 IDAAYIQLIYPV; SEQ ID NO. 43LIYPVTNFQKHM SEQ ID NO. 44 Between Ig-like Aa 209-240 LEENKPTRPV; domain2 and 3 SEQ ID NO. 45 (Hinge) NKPTRPVIVS SEQ ID NO. 46 Ig-like 2 loope2-f2 Aa 181-200 VAEKHRGNYT; (pas int.ligand) SEQ ID NO. 47 WNGSVIDEDSEQ ID NO. 48 IL-1RacP Juxtamembranous Aa 330-370 VPAPRYTVEL; SEQ ID NO.49 APRYTVELA; SEQ ID NO. 50 Hinge regions: Loop Ig-1-2: Aa 115-160VQKDSCFNSPM; SEQ ID NO. 51 MKLPVHKLY SEQ ID NO. 52 Loop Ig-2-3 Aa170-266 VGSPKNAVPPV; SEQ ID NO. 53 VTYPENGRTF; SEQ ID NO. 54 IHSPNDHVVY;SEQ ID NO. 55 dimerization region Aa 200-215; LISNNGNYT; 275-295; SEQ IDNO. 56 300-315 VWWTIDGKKPD; SEQ ID NO. 57 WTIDGKKPDDI; SEQ ID NO. 58HSRTEDETRTQ SEQ ID NO. 59

Cytokines receptors modulators according to specific embodiments of thepresent invention will now be described as well as the procedure toidentify them and to test their efficiency in vitro and/or in vivo bythe following non-limiting examples.

Example 1 VEGFR Identification of VEGFR2 Antagonists

VEGF is a proliferating agent for endothelial cells. Its receptor(VEGFR) is present at the plasma membrane of endothelial cells as amonomer and its homodimerization is necessary for generatingautophosphorylation via its intrinsic tyrosine kinase domain.

The method of identifying VEGFR antagonists of the present invention isbased on the localization of extracellular flexible regions includingregions between domains and juxtamembranous regions of the receptor thatare important for the appropriate conformation and oligomerization ofthe subunits of the receptor and its resulting activation. These regionswere established based on crystal structure data provided bycrystallography. The antagonists able to bind to these regions block thesignal transduction by interfering with the oligomerization. The regionsso identified appear in green in FIG. 3. One of those regions is locatedunder the IG-like 3 domain where ligand binding is located, namelybetween residues 320 and 350. The ligand binding location also appearsin FIG. 1. A second region was identified in the oligomerization domainof two subunits of Ig-like 4, namely between residues 350 and 400. Athird region was identified located at the juncture of the receptor withthe cellular membrane, namely between residues 745 and 770. This regionis important for the dimer stability. These regions do not interferewith the ligand binding so that any antagonist (peptide, small molecule)targeting these regions is not a competitor for the ligand binding sites(non-competitive antagonist) and prevents or limits the oligomerizationrequired for the autophosphorylation of the receptor. Three D-peptidesof up to 12 amino-acids (designated 2.1, 2.2 and 2.3) were derived fromthe amino-acid sequence of these regions and tested as antagonists. Asmentioned earlier, D-peptides are preferred over subfragment peptides(of course subfragments could also be rendered protease resistant bywell known means) because they are less likely degradable by variousproteases. These particular peptides were selected among all those thatcould have been derived from the identified flexible regions of interestbecause of their specificity to VEGFR-flk-1: sequences alignments wereperformed with other receptors from VEGFR's family (PDGFR, Flt-1)showing the specificity of the selected three peptides. Of course, suchalignments enable a selection of other specific peptides oralternatively of more general antagonists. It should be understood thatthe principles related to positioning discussed herein in relation toVEGFR can be applied to other types of cytokine receptors sharingsimilar morphologies.

The location of the three peptides appear in FIG. 1, the ligand bindingregion appears in red, the oligomerization domain per se appears ingreen and the tyrosine kinase domain appears in purple.

In FIG. 3, the domains of the VEGFR isoform VEGFR-2 are identified witharrows pointing at the start of each domain. The regions whereantagonists of the present invention may bind to prevent theoligomerization and/or activation of the receptor are boxed orunderlined. The underlined sequences denote the regions between domainswhile the boxed sequences denote the juxtamembranous regions. Theregions from where peptides 2.1, 2.2 and 2.3 are derived are identifiedin italic and are underlined. The sequences that the peptides targetaccording to the invention appear underlined and boxed.

Characterization of Peptides In Vitro

To determine the efficient and non cytotoxic concentration of VEGF touse in the assay, a dose-response curve of VEGF was generated in twotypes of cells, namely microvascular endothelial cells and pulmonaryartery endothelial cells (PAEC) that had been transfected with the Flk-1gene. The proliferation was then measured in those two types of cells inthe presence of peptides 2.1, 2.2 and 2.3 and of VEGF (2 ng/ml) pursuantto the incorporated tritiated thymidine method. The cells werepreincubated at 37° C. with the different peptides at differentconcentrations. They were incubated with VEGF (2 ng/ml) for 24 hours.The cells were contacted with ³H-Thymidine for 24 hours, washed andlysed. The radioactivity was measured with a scintillation counter.

As may be seen in panels A and B of FIG. 2, the peptides 2.1, and 2.2completely abrogated VEGF induced proliferation in microvascularendothelial cells, and in PAEC with an EC₅₀ of 9 μM, respectively. Inaddition, using these PAEC transfected with the cDNAs for either of theVEGFR isoforms Flk-1 and Flt, the selectivity of the peptides wasdemonstrated as they were shown to be ineffective in modulatingbiological functions in the VEGFR Flt isoform-containing cells (data notshown).

Characterization of Peptides In Vivo Ischemic Retinopathy Model

The efficiency of the selected peptides was verified in vivo in aischemic retinopathy model, a phenomena highly dependant on VEGFactivation. Rat pups were exposed to 80% O₂ followed by a period ofnormoxia (21% O₂). The peptides were injected at a final concentrationof 10 μM in the vitreous body. The retinas were then retrieved, coloredwith the ADPase method and mounted on slides. Photographs of the retinaswere taken with a microscope linked to a computer and the vasculardensity was evaluated with the Image prosoftware. As illustrated in FIG.2, panel C, the results of this experiment demonstrated that allpeptides tested prevented induced neovascularization in vivo. Peptide2.2 was shown to be the most effective inhibitor of neovascularization.Specific peptides of the present invention were shown to prevent effectsgenerated by activation of Flk-1 with VEGF by interfering with flexibleregions of Flk-1 receptor.

Example 2 Insulin-Like Growth Factor-1 Receptor (IGF-1R)

IGF-1 is a small peptide and a member of a family of insulin relatedpeptides. It consists of 70 amino acids and has structural similaritywith insulin. IGF-1 is secreted by many tissues (cartilage, bone,epithelium, endothelium) but mostly by the liver to act on other tissuesin an endocrine fashion. It exerts its actions by binding to IGF-1R uponwhich it sends a mitogenic signal. It can also protect cells fromapoptosis, promote proliferation, regulate cell adhesion and motilityand differentiation. The receptor itself is expressed in most cell typesexcept in the hepatocytes. Because of its growth inducing functions,IGF-1R is also very much involved in malignant transformation ordifferentiation in various types of cancer such as glioblastomas,neuroblastomas, prostate, breast and ovarian cancer.

IGF-1 plays a critical role in cell growth, survival and metastaticdifferentiation. IGF-1R is a transmembrane tyrosine kinase protein,which is widely expressed. Increased IGF-1 R expression is observed in anumber of tumour types, and epidemiological data implicates it incancers such as those of the prostate and breast. Recent progress hasbeen made on its 3-dimensional structure.

Design of Peptides for IGF-1

The approach described in Example 1 is used to generate antagonists toIGF-1R. The precise localization of these regions is described in Table1 above along with exemplary sequences of subfragment peptides ormodified peptides targeting one of these regions and presentingspecificity to IGF-1R. Three D-peptides (designated APG201, APG202 andAPG204) were then derived from the amino-acid sequence of these regionsto act as antagonists. The sequences of these peptides antagonists areas follows: APG-201 SLFVPRPERK; APG-202 ESDVLHFTST; APG-204 LRKYADGTL.They generally correspond to the subfragment peptides having sequencesSEQ ID NOs: 11, 14 and 16, respectively, except where the subfragmentpeptide contained an isoleucine. Similarly, In that case, thisamino-acid was replaced by leucine in the synthesized peptide foreconomic reasons.

Characterization of Peptides In Vitro and In Vivo

The affinity is determined using binding studies on cells expressing andoverexpressing IGF-1R. The selectivity is tested by performing bioassayson cells expressing receptors from the same family as IGF-1R and thespecificity is tested against receptors of another family of cytokine.

The proliferation of IGF-1 was measured in A549 carcinoma cells in thepresence of peptides APG201; APG202 and APG204 and of IGF-1 (10ng/ml-Panel A) and (1 ng/ml-Panel B) pursuant to the incorporatedtritiated thymidine method. The cells were preincubated at 37° C. withthe different peptides at different concentrations, namely 10⁻⁷, 10⁻⁶and 10⁻⁵M. They were then incubated with IGF-1 (10 ng/ml or 1 ng/ml) for24 hours. The cells were then contacted with ³H-Thymidine for 24 hours,washed and lysed. The radioactivity was then measured with ascintillation counter.

As may be seen in panels A and B of FIG. 8, the peptides completelyabrogated IGF-1 induced proliferation in A549 carcinoma cells with anEC₅₀ of 10⁻⁸M for APG-202 and 204; and of 10⁻⁶M for APG-201.

Further in vitro testing of the antagonists are conducted as describedin Table 2.

In vivo experiments are described in Table 5.

TABLE 2 In vitro bioassays for IGF-1R antagonist screening Cells TypeBioassay Method Du145 Prostate cancer Proliferation ³H-Thymidine cellline Akt incorporation phosphorylation Western Blot PC12 Pheochromo-Same as above Same as above cytoma cell line

Example 3 Interleukin 4 (IL-4)

IL-4 is a key cytokine involved in the development of allergicinflammation and allergy. It is generated early on in the process ofinflammation in asthma. In allergy it is associated with the productionof IgE immunoglobulins by B lymphocytes and will also up-regulate theexpression of the IgE receptor on cell surface of B-lymphocytes,basophils and mast cells. In asthma it induces the expression ofvascular cell adhesion molecule (VCAM-1) on vascular endothelium. Thiseffect leads to direct migration chemotaxis of T lymphocytes, monocytes,basophils and eosinophils to the inflammatory site on pulmonary vascularendothelial cells. IL-4 inhibits eosinophil apoptosis and promoteseosinophilic inflammation by augmenting their presence in part byincreasing expression of eotaxin. Another essential biological effect ofIL-4 is Th2 differentiation and proliferation; in this process IL-4diminishes T lymphocyte apoptosis. The II-4 receptor is a cell-surfaceprotein consisting of an α subunit coupled to a γ subunit for signaltransduction; its activation requires oligomerization.

Although IL-4R and IL-13R share a similar IL-4Rα chain, the tworeceptors exhibit distinct functions; moreover, the main receptorpresent on TH2 cells is that of IL-4, which for the most part consistsof the IL-4Rα and IL-4γc chains. Nevertheless, the identification ofmodulators of IL-4R activity. Derived from the IL-4Rα are expected toalso modulate IL-13R activity.

Design of Peptides for IL-4R

The approach described in Example 1 is used to generate antagonists toIL-4R. The precise localization of these regions is described in Table 1above along with exemplary sequences of subfragment peptides or modifiedpeptides targeting one of these regions and presenting specificity toIL-4R

Characterization of Peptides In Vitro and In Vivo

The affinity is determined using binding studies on cells expressing andoverexpressing IL-4R. The selectivity is tested by performing bioassayson cells expressing receptors from the same family as IL-4R and thespecificity is tested against receptors of another family of cytokine.

The proliferation of IL-4 was measured in A549 carcinoma cells in thepresence of peptides API-401, API-402, API-403, API-404 and API-405 andof IL-4 (1 ng/ml) pursuant to the incorporated tritiated thymidinemethod. The cells were preincubated at 37° C. with the differentpeptides. They were then incubated with IL-4 (1 ng/ml) for 24 hours. Thecells were then contacted with ³H-Thymidine for 24 hours, washed andlysed. The radioactivity was then measured with a scintillation counter.The sequences of peptides antagonists used are as follows: API-401YREPFEQHLL, API-402 SDTLLLTWS; API-403 LYNVTYLE; API-404 LAASTLKSGLS;and API-405 KPSEHVKPR. They generally correspond to the subfragmentpeptides having sequences SEQ ID NOs: 35, 36, 37, 38 and 39,respectively except where the subfragment peptide contained anisoleucine. In that case, this amino-acid was replaced by leucine in thesynthesized peptide as mentioned previously.

As may be seen in FIG. 10, four out of five peptides prevented IL-4 fromstopping proliferation in A549 carcinoma cells.

Further In vitro testing of the antagonists is conducted, as describedin Table 3. In vivo experiments are described in Table 5.

TABLE 3 In vitro bioassays for IL-4R antagonist screening Cells TypeBioassay Method T helper T helper cells Proliferation ³H-Thymidine Aktincorporation phosphorylation Western Blot PAEC Human pulmonary VCAM-1expression Western blot artery endothelial cells

Example 4 Interleukin-1

Interleukin-1 (IL-1) plays a primary upstream role in the regulation ofinflammation by stimulating generation of other inflammatory mediatorsand by enhancing the process of inflammation directly. Along with TNF,IL-1 is considered as a prototype for inflammatory cytokines. Theeffects of IL-1 are not limited to inflammation and this cytokine playsa role in bone formation and remodeling, insulin secretion and feverinduction. IL-1 is also a major player in acute and chronic inflammation(e.g. septic shock, inflammatory bowel diseases, osteoarthritis, orrhumatoid arthritis), Alzheimer's disease and a number of autoimmunediseases. Monocytes are predominant sources of IL-1 but many other celltypes express the protein: non-limiting examples include fibroblasts,endothelial cells, smooth muscle cells, osteoclasts, astrocytes,epithelial cells T-cells, B-cells and numerous cancer cells.

The interleukin-1 family of proteins consists of distinct butstructurally related molecules: IL-1α, IL-1β and IL-18 which elicit abiologic response and IL-1ra, a naturally produced receptor antagonist.IL-1α is the predominant form in mice, IL-1β is predominant in human butboth exert their effect through the same receptor. In addition, IL-1induces the production of other inflammatory mediators like IL-6 andprostaglandin PGE₂ (induces COX-2 and PGE synthase expression) andinduces proliferation and activation of numerous cell types.

As a major pro-inflammatory cytokine, IL-1 is a potentially powerfultarget for therapeutic interventions in diseases associated witharticular cartilage injury such as in arthritis. Osteoarthritis andrhumatoid arthritis are only second to heart diseases for causing workdisabilities in USA and their prevalence increase dramatically with age.Approximately 60 millions of American >40 years of age are at risk. In1997, direct medical and disability costs for arthritis wereapproximately $75B (US). Other important disorders for which IL-1contributes significantly include ulcerative colitis and Crohn'sdisease, which are also major causes of absenteeism in USA, and othertypes of auto-immune diseases.

Two distinct receptors of IL-1 have been cloned and characterized: IL-1Rwhich generates the biological effects of IL-1, and IL-1RII which is anatural antagonist. In addition, a receptor accessory protein(IL-1RAcP), which is the putative signal-transducing subunit of thereceptor complex has been identified. IL-1R type I is found mainly on Tcells, keratinocytes, fibroblasts, chondrocytes, synoviocytes andepithelial cells. In order to generate a biological effect, IL-1R has tobind to IL-1 and subsequently to IL-1 RacP which is necessary for signaltransduction. The extracellular portion of IL-1R contains 3 Ig-likedomains that bind IL-1. Of note, according to studies involvingantibodies directed against extracellular portions of IL-1 RacP, thelatter does not interact with the cytokine and could therefore also bean excellent target for non-competitive peptidomimetic design.

Design of Peptides for IL-1R

The regions of the IL-1 receptor complex which were targeted are thethird domain of IL-1R containing a flexible region and interacts withthe accessory protein but not with the ligand. The equivalent domain onIL-1 RacP, is the juxtamembranous regions of IL-1R and IL-1AcP and theregions between the second and third extracellular domains of IL-1 RacP.The precise localization of these regions is described in Table 1 abovealong with exemplary sequences of subfragment peptides or modifiedpeptides targeting one of these regions and presenting specificity toIL-1R.

Characterization of Peptides In Vitro and In Vivo

The affinity of the subfragment peptides or derivative is determinedusing binding studies on cells expressing or overexpressing IL-1R. Theselectivity is tested by performing bioassays on cells expressingreceptors from the same family as IL-1R (e.g. IL-18R) and thespecificity is tested against receptors of another family of cytokine.

The proliferation effect of IL-1 was measured in A549 carcinoma cells inthe presence of peptides API101; API103 and API106 and of IL-1 (10ng/ml-Panel A) and (1 ng/ml-Panel B) pursuant to the incorporatedtritiated thymidine method. The cells were preincubated at 37° C. withthe different peptides at different concentrations, namely 10⁻⁶, 10⁻⁵and 10⁴M. They were then incubated with IL-1 (10 ng/ml or 1 ng/ml) for24 hours. The cells were then contacted with ³H-Thymidine for 24 hours,washed and lysed. The radioactivity was then measured with ascintillation counter. The sequences of the peptides antagonists usedare as follows: API-101 APRYTVELA, API-103 MKLPVHKLY; and API-106VGSPKNAVPPV. They generally correspond to the subfragment peptideshaving sequences SEQ ID NO: 50, NO: 52 and NO: 53, respectively exceptwhere the subfragment peptide contained an isoleucine. In that case,this isoleucine was replaced by a conservative leucine in thesynthesized peptide for economic reasons.

As may be seen in panels A and B of FIG. 9, the peptides completelyabrogated IL-1 induced proliferation in A549 carcinoma cells with anEC₅₀ of 10⁻⁶M for API-101 and 103; and of 10⁻⁵M for API-106.

The goal of the next experiment was to verify if the identified peptidescan reverse the physiological actions of the natural cytokine in vivoeither by injecting them through the jugular or directly in the stomach(to verify the stability of the peptide through the digestive tractus).300 g Sprague-Dawley rats were anesthetized with isoflurane (2.54%).IL-1β was injected through the jugular. Blood was taken from the carotidfor further analyses before and after (10 minutes) every injection.Peptides were then injected either directly in the stomach with acatheter or in the jugular at the concentration desired. Arterial bloodpressure and other physiological characteristics were monitored at alltime.

A severe hypotension induced by IL-1 was observed when administrated tothe rats by either ways mentioned above. The following peptidesconstitute examples of antagonists that were able to preventhypotension:

API-101.10 (target: juxtamembranous portion of the accessory protein ofIL-1R, derivative of API-101):

-   -   1) When administrated by jugular injection after IL-1β injection        (5 ug/kg) it prevented hypotension by 95% at a concentration of        10⁻⁸M. This demonstrated that the peptide has an hypotensor        effect in vivo in animals by reversing the effect of IL-1β (data        not shown).    -   2) When administrated directly into the stomach, the peptide at        a concentration of 10⁻⁵M, reduced IL-1β induced hypotension by        60%. This result demonstrated that oral administration of the        101.10 peptide still maintained a major effect on IL-1β induced        hypotension. (Data not Shown)

In another experiment, vasomotricity variation of piglets pial vesselswas studied to further evaluate the particular effect of cytokinereceptor subfragments on the vasodilatator effect of IL-1β. Brains weredissected from Yorkshire piglets. Slices of brain exposing the pialvessels were pinned to a wax base of a 20 ml bath containing Krebsbuffer (pH 7.4) equilibrated with 95% O₂-5% CO₂ and maintained at 37° C.Microvessels were visualized and recorded using a video camera mountedon a dissecting microscope. Vascular diameter was measured using adigital image analyzer and the images were recorded before and aftertopical application of constricting agent U46619 at 10⁻⁷M. Afterstabilization of the vasomotricity, IL-1β was added until stabilizationof vasodilatation. Peptides were then injected at differentconcentrations from 10⁻¹⁰ to 10⁻⁵M. Reversal of vasodilatation (i.e.vasoconstriction) was visualized and measured as previously mentioned.IL-1β induced vasodilatation in the microvasculature of the piglet brainwas observed. Examples of the inhibitory activity of cytokinesubfragment peptides are given below:

-   -   1) API-101 and 101.10 (Juxtamembranous part of accessory        protein) could prevent the vasodilation induced by IL-1β (75        ng/ml) with an IC₅₀ of 182 nM (API-101) and 10.8 nM        (API-101.10). The range of concentrations of the peptide        administered was from 10⁻¹⁰ to 10⁻⁵M (data not shown)    -   2) API-108 (hinge Ig-3 region of accessory protein) could        prevent vasodilatation with an IC₅₀ of 1.9 nM (data not shown).        The range of concentrations of the peptide administered was from        10⁻¹⁰ to 10⁻⁵M.

These results demonstrate that targeting of two flexible regions of onecomponent of the receptor we could prevent IL-1β activity at a very lowIC₅₀ and therefore with a very high efficiency.

Another way of assessing the effect of cytokine receptor subfragments onIL-1R activity in vivo is by measuring PGE₂ levels in rat blood serum.Rat blood samples were collected from in vivo experiments (e.g. Protocolfor IL-1 induced hypotension) and centrifuged at maximum speed for 15minutes. The serum was then passed through a Waters column in order toisolate the lipidic part. Samples were evaporated and PGE₂ quantitieswere determined with an RIA assay using a commercial kit (Cederlane).

If the cytokine receptor subfragment peptides can prevent hypotention invivo they should be able to prevent also the synthesis of PGE₂. Theprostaglandin was therefore measured in serum of rats used forexperiments mentioned above (e.g. Arterial Blood Pressure variationmeasurement). An example of results obtained with a particular cytokinereceptor subfragment peptide is described below:

-   -   1) API-101.10 could prevent PGE₂ synthesis in vivo by 80% when        the peptide was injected in the jugular. The same results were        obtained when the peptide was injected directly in the stomach        (data not shown).

These experiments demonstrate that the identified peptides derived fromdifferent flexible regions of a cytokine receptor (in this particularexample, receptor IL-1R/IL-1 RacP) are efficient and very potent invitro and an vivo at reversing various biological effects of IL-1β.

From these experiments the efficiency and specificity of the method usedto select particular cytokine subfragment peptides to modulate cytokinereceptor activity is clearly demonstrated. Furthermore, the particularexperiments presented above (with the IL-1R/IL-1RacP receptors) servesas a complete example of how one can select a particular cytokinereceptor subfragment peptide (derivitize and/or protect it if desired),test its modulating activity in vitro and than its efficiency andpotency in vivo. It also demonstrate that the modulating activitiesdemonstrated in vitro are translatable to the in vivo situation.

The stability and selectivity of the peptides in vitro and in vivo isfurther verified with the tests described in Table 4, and Table 5 below,respectively.

TABLE 4 In vitro bioassays for IL-1R antagonist screening Cells TypeBioassay Method Chondrocytes Human PGE₂ levels RIA kit chondrocytes IL-6RIA kit Proliferation ³H-Thymidine Collagenase incorporation expressionWestern Blot RPE Human retinal Same as above Same as above pigmentepithelial cells Thymocytes EL4-Mouse Proliferation ³H-Thymidinethymocytes-High incorporation IL1R expression Fibroblasts Human F7100Proliferation ³H-Thymidine incorporation

Table 5 summarizes the nature of the in vivo experiments performed withvarious peptides of the present invention. They are presented in moredetails below.

TABLE 5 In vivo experiments to assess efficacy and specificity ofantagonists against IL-1R, IGF-1R and IL-4R Target Animal model MethodTreatment Parameters IL-1R Collagen-induced arthritis in s.c. injectionsFollowing onset of Destruction of rat of type II arthritis, cartilagecollagen in continuous assessed by incomplete delivery of thehistological Freund's drug via osmotic staining and adjuvant pumpdigital imaging Arterial blood pressure Injection of 10 minutes Bloodpressure variation measurement in IL-1b in following IL-1b, variationrats jgular injection of peptide measurements antagonist in jugular orstomach. Vasomotricity experiment on Topical Following U46619 Vascularpiglet pial vessels application of induced diameters U46619vasodilatation, agent as application of vasoconstrictor peptideantagonist than, IL- in microvessels 1b as a vasodilatator PGE₂ levelsin rat blood Injection of injection of peptide PGE₂ levels serum IL-1bin antagonist in jugular jugular or stomach and measurement of PGE₂levels by RIA kit Acute septic shock in rat LPS-induced Preceding i.v.Blood pressure, septic shock bolus of LPS the body temperature animalwill receive and cardiac an i.v. bolus of the rhythm will be antagonistmonitored during the whole experiment (60 min) IGF-IR Tumor growth ins.c. injection Continuous Tumor size immunosuppressed mouse of tumoraldelivery of the monitoring (nude mouse) cell line antagonist withosmotic pump after latency to obtain solid tumor IL-4R Sensitization ofthe airways Exposure of i.p. injection of IgE and TNF-γ in newborn micethe animals receptor dosage ovalbumin antagonist (i.p. injection andaerosolized)

Acute Septic Shock in Rats

The efficiency of the peptides is also verified with the acute septicshock in Sprague-Dawley rat. Sprague-Dawley (160-180 gm) rats (CharlesRiver) are anesthesed with a solution 9:1 xelazine/ketamine at aconcentration of 1 mg/Kg. A tracheotomy is performed so as to maintainventilation with a tube linked to a respirator. A cannula is insertedinto the right carotide artery to enable monitoring of the systemicarterial with a Stratham pressure transducer linked to a multichannelGould apparatus. The right jugular vein is cannuled to enable drugadministration. The animal is placed under radial heat to maintain aconstant normal temperature. The septic shock is obtained by systemicinjection of a lipopolysaccharide bolus (LPS) (1 mg/kg: Sigma). Adecrease of about 30 mm Hg is observed after ˜5 minutes.

Collagen-Induced Arthritis Protocol in Lewis Rat

Type II Collagen (CII) that has been isolated and purified from bovinearticulary is obtained from Sigma. CII (2 mg/ml) is dissolved over nightat 4° C. with agitation in 0.01 M acetic acid. The solution is thenemulsified in an incomplete Freund's adjuvant (CII: ICFA, DifcoLaboratories, Detroit, Mich.). Lewis female rats (Charles River) of140-180 gm and of 8 week old are immunised with 0.5 ml of the emulsion(0.5 mg CII) with many intradermal injections in the back and one or twoinjections in the tail base. The animals are then reinjected 7 dayslater in the tail base with 0.2 ml (0.2 mg CII) so as to obtain an acuteinflammatory reaction. A different time points during the experiment (1to 24 days) animals are sacrificed and knuckle joints samples are takento be fixed and coated so as to enable cryosections of 6-7 μm. A doublecoloration of Goldner and toluidine blue is performed on slides tomeasure the importance of the articular inflammation. Digitalised imagesare taken and analysed with the Image Pro Plus™ 4.1 software.

Tumor Growth in Immunosuppressed Mouse (Nude Mouse)

The colon Colo 205™ carcinoma cell line is obtained from the AmericanType Culture Collection (ATCC: Rockville, Md.). Cells are maintained ina RPMI-1640 culture- and grown in 100 mm Petri at 37° C. in a humidifiedatmosphere controlled to maintain 5% CO₂ and 95% air. The medium issupplemented with 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin and 100μg/ml streptomycin.

2.5×10⁶ carcinoma colon Colo 205™ cells in 100 μl de PBS are injectedsubcutaneously in the back (needle 25 G:BD, NJ) in 6 weeks oldimmunodeficient female mice (Balb/c, nu/nu: Charles River). Treatmentbegins 5 days after injection of tumorous cells measuring ˜0.5×0.5 cm.the tumour volume is measured every two days according to the followingformula: length×width×height, with a vernier caliper. 14 days after thebeginning of treatments, animals are sacrificed and tumours are sampledto be weighted and measured in volume. Specimens are then fixed in a 10%formalin buffer for 24H and then transferred in 70% ethanol. Tumours arethen coated with paraffin and sections are cut for immunohistochemistrypurposes. The general morphology is evaluated with a hematoxyline/eosincoloration.

Although the present invention has been described hereinabove by way ofspecific embodiments thereof, it can be modified, without departing fromthe spirit and nature of the subject invention as defined in theappended claims. In particular, although the flexible regions of allcytokines have not all been described herein nor have all peptidicextracellular non competitive modulators encompassed by the presentinvention targeting these regions have been described, in light of theprocedure described above for screening peptides and identifyingpeptides of the present invention, a person of ordinary skill in the artwould be able to rapidly develop peptidic modulators of cytokinereceptor by selecting peptides of 5 to 20 amino acid derived from knownflexible regions of cytokines.

REFERENCES

-   Christine Piossek et al. “Vascular Endothelial Growth Factor (VEGF)    Receptor II-derived Peptides Inhibit VEGF” The Journal of Biological    Chemistry vol. 274, No. 9, Feb. 26, 1999, pp. 5612-5619.-   Daren C. W. Tan et al “A small peptide derived from fit-1 (VEGFR-1)    functions as an angiogenic inhibitor” FEBS Letters 494 (2001)    150-156.-   Guy Vigers et al. “X-Ray Crystal Structure of a Small Antagonist    Peptide Bound to Interleukin-1 Receptor Type 1” The Journal of    Biological Chemistry vol. 275, No. 47, Nov. 24, 2000, pp.    36927-36933.-   Do-Young Yoon et al. “Antibodies to domains II and III of the IL-1    Receptor Accessory Protein Inhibit IL-1β Activity But Not Binding:    Regulation of IL-1 Responses Is Via Type I Receptor, Not the    Accessory Protein” Journal of Immunology, 1998.

1. A method for identifying a non-competitive peptide which inhibits theactivity of a cytokine receptor, said method comprising selecting acandidate peptide containing from about 7 to about 20 amino acidsderived from a flexible region of the receptor, and determining theability of the candidate peptide to inhibit the oligomerization and/oractivity of the receptor by measuring a biological activity of saidreceptor in the absence or presence of the candidate peptide, wherein anon-competitive peptide is selected when the activity of the receptor ismeasurably lower in the presence of the candidate peptide as compared toin the absence thereof. 2-37. (canceled)